Program Director/Principallnvestigator (Last, First, First, Middle): Crabtree, Gerald 2 R01 Program Director/Principal Investigator (Last, Middle) : Crabtree , Gerald 2 ROl E-, A1060037-06 AI060037 -06 shape repertoire lymphocytes has been one the most The mechanisms that shape the repertoire of T lymphocytes has been one of the most enduring mysteries in immunology. The process of repertoire selection is critical to the enduring mysteries in immunology. The process of repertoire selection is critical to the development of effective immunity to foreign antigens, transplant rejection, antigens, transplant rejection, immunosuppression and also to the pathogenesis of autoimmune disease. Substantial Substantial immunosuppression and also the autoimmune evidence mechanism in which signals the antigen experimental evidence supports a mechanism in which intense signals at the antigen receptor lead to the elimination of clones of T cells bearing receptors binding self to elimination cells bearing receptors binding antigen, while weaker signals mediated by TCR's that match MHC molecules lead to the lead to antigen, mediated cells able to mount immune responses to foreign antigen survival and differentiation of T cells able to mount immune responses to foreign antigen. . distinguished and These observations indicate that signals of different intensity must be distinguished and sorted to produce distinct cell fates. The problem of discriminating signals of different problem sorted to produce distinct cell fates. signals different unsolved question in biology and evidence indicates that signal intensity is a general and unsolved question in biology and evidence indicates that signal intensity at several different morphogen receptors also determines cell fate. Lymphocyte receptors also determines cell fate. Lymphocyte development is well suited to solve this general problem as biochemical, cell biologic well suited this general problem biochemical, cell and genetic approaches can all be effectively combined. Recent evidence indicates that can combined. indicates negative selection in response to intense signals at the antigen receptor is mediated by signals mediated by the transcriptional activation of BIM by a Signaling pathway requiring PKCa. In contrast, BIM In contrast, transcriptional activation signaling pathway requiring positive selection appears to arise when early signaling by calcineurin leads to a brief selection when signaling by calcineurin leads to period of ERK hypersensitivity allowing differential responses to weak signals at the allowing differential responses to signals the alpha/betaTCR in CD4/CD8 T cells or Erk alpha/betaTCRin CD4+/CD8+ T cells.. Mutations in components of the calcineurin or Erk pathways produce selective defects in positive selection with no apparent defect in selective in selection with in negative selection, while mutation of Bim leads to selective defects in negative selection of Bim leads to selective defects in negative selection with no defect in positive selection . These observations indicate that the mechanisms with no in positive selection. These observations indicate that the mechanisms sort signals to distinct fates in T cells must operate upstream of Bim, that operate to sort signals to distinct fates in T cells must operate upstream of Bim, Calcineurin and Erk, yet downstream of CD3, LAT, VAV and other molecules required of CD3, LAT, VAV and other molecules required Calcineurin for both positive and negative selection . We have revised our plans to focus on the and negative selection. We have revised focus on the comments of the reviewers or our application . Hence we will focus on the strong points comments the reviewers application. Hence we will focus on the strong points including using a genetic strategy to identify the targets of calcineurin signaling in CD4 including using genetic strategy to identify the targets of calcineurin signaling in CD4 ICD8- thymocytes that modulates Erk activity and are essential for positive selection.. /CD8 thymocytes that modulates Erk activity and are essential for positive selection be first with deletion The genes identified will be tested 'first with simple RNAi methods and later with deletion to define their role in T cell development. As the reviewers suggested we will document cell development. reviewers suggested will the role of signaling pathway between PKCa and BIM using additional models of between on negative selection based on other antigens and also as the reviewers suggested focus and also reviewers suggested focus on using a "on-time" deletion model of negative selection. Proteomic methods will be "on-time" deletion model negative selection. Proteomic methods will be in bim regulators dependent upon negative selection used to define modifications in the bim regulators dependent upon negative selection of signals, ultimately mediated by PKCci. Finally we will complete the construction of the signals, ultimately by PKCa . Finally Positive Selection Indicator mice that will be essential for the genetic strategies Positive Selection will be essential the strategies described in the original application. Our revised studies will set the ground to define the Our revised studies will set the ground to define the described in original elements in a pathway carrying signals from the antigen receptor and PKCcx to the carrying signals from the antigen receptor and PKCa to the proteins controlling BIM activation and negative selection. Precisely defining the full proteins controlling BIM activation and negative selection. 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